I propose to utilize basic tissue engineering principles and high throughput methods to regenerate cartilage. My hypothesis is that human embryonic stem (hES) cells can produce functioning cartilage that can be integrated in vivo. I will attempt to develop a microfluidic polymer-cell culture array that can simultaneously select the best combination of scaffold material, growth factor(s) and gene(s) for cartilage regeneration. As part of my research plan, I will first determine if this system can be used to identify best conditions to differentiate stem cells into chondrocytes. Using these results, I will scale up the construct into 3D scaffolds that can release the selected growth factors and genes, and ascertain which conditions can stimulate hES cells to produce cartilage in vitro. Finally, this construct will be evaluated in vivo for its chondrogenic potential. My specific aims are: Aim 1. Develop a high throughput system for simulating complex stem cell environments. Initially, rapid screening techniques for cell-polymer interactions will be used to select for polymers most favorable to hES cell attachment. Then, hES cell differentiation into chondrocytes will be evaluated on selected materials in combination with various growth factors and genes using a microfluidic polymer-cell culture array. Chondrocyte differentiation will be assessed with immunohistochemistry for DMA, type II collagen and glycosaminoglycans (GAGs). Aim 2. Scale up most promising candidates from Aim 1 for in vitro hES cell studies. 3D scaffolds containing selected growth factors and genes will be fabricated and ranked according to their ability to differentiate hES cells into chondrocytes and to produce cartilage. Polymer-hES cell constructs will be examined for cell viability, DMA, type II collagen and GAG content, gene activity, compressive modulus, and histological phenotype and organization. Aim 3. Evaluate in vivo performance of the two best scaffold systems selected in Aim 2. The 3D scaffolds loaded with the most promising growth factors and genes will be seeded with hES cells. First, the constructs will be evaluated subcutaneously in rats to test their ability to differentiate hES cells and synthesize cartilage. Secondly, they will be evaluated in a knee cartilage model, and assessed for their ability to differentiate hES cells, stimulate matrix production and integrate with native tissue. In the United States, osteoarthritis has a prevalence of 43 million people, most of whom are over 65 years of age. The results of this work will lead to significant progress toward regenerating human cartilage, thereby improving the qualty of life for many Americans.